Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 1 Differential expression of AP1 factors and aromatase in human granulosa cells in response to forskolin (FSK) treatment. (A) KGN cells were treated with forskolin (25 mM) and were harvested at the time intervals indicated on top of the figures. Total RNA was isolated, reverse transcribed, and analysed by semi- quantitative PCR using primers specific for cJun, JunD, JunB, cFos, Fra2, aromatase, and b-actin (as an internal control). (B) Cell lysates were prepared at different intervals after forskolin treatment and an equal amount of total proteins were resolved by SDS– PAGE. Immunoblotting was performed using specific antibodies. a-Tubulin was used as a loading control. Results are representative of two experiments. The arrows in (b) and (d) indicate the specific bands for JunD and aromatase, respectively

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Quantitative Proteomics, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, SDS Page, Western Blot

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 2 Ectopic expression of Jun proteins in KGN cells inhibits transcription from the ovary-specific promoter of the aromatase gene (PII). (a) KGN cells were transfected with the PII-luciferase reporter construct and Jun or empty expression vector (pcDNA3). Cells were then incubated in the absence (open bars) or presence (solid bars) of forskolin (25 mM). An anti-Flag immunblot of the tagged Jun proteins is shown in the inset. (b) JunB inhibits the cAMP-dependent transcription from the PII promoter in a concentration-dependent manner. KGN cells were co-transfected with the PII-luciferase reporter construct and increasing amounts of the JunB expression construct (50, 100, 250 ng). The total amount of expression vector in each sample was maintained constant by using the pcDNA3 empty vector. Cells were then incubated in the presence or absence of forskolin (25 mM) overnight before harvest. Luciferase data shown are average of results from triplicates

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Expressing, Transfection, Luciferase, Construct, Plasmid Preparation, Incubation, Concentration Assay

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 3 Jun proteins repress transcription in a promoter-specific manner. (a) Luciferase reporter plasmids harboring synthetic AP1- responsive, collagenase and cyclin D1 promoters were co-trans- fected into KGN cells with the JunB expression construct. (b) JunB and cJun expression constructs were transfected in KGN cells along with either the aromatase PII-luciferase or (151/1) StAR- luciferase reporter construct. Following transfection, cells were treated with or without forskolin (25 mM) overnight and luciferase activity in cell lysates was determined. Luciferase data shown are average of results from triplicates

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Luciferase, Expressing, Construct, Transfection, Activity Assay

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 4 Repression of endogenous aromatase expression by ectopic JunB. KGN cells were infected with recombinant adeno- virus that expressed either LacZ (control) or JunB at the indicated MOIs for 36 h, followed by treatment with forskolin (25 mM) for 4 h. Cells were harvested 40 h post infection and analysed by immunoblotting for the protein levels of JunB (a) and aromatase (b). (c) KGN cells were infected with 200 MOI of adeno-LacZ or adeno-JunB for 36 h, and subsequently treated with forskolin (25 mM) for 2 and 4 h. Total RNA was prepared and used in semiquantitative PCR for the measurement of the aromatase mRNA level. b-Actin was used as an internal control in both protein and mRNA analyses. Results are representatives of two separate experiments

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Expressing, Infection, Recombinant, Virus, Control, Western Blot

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 5 TAM-67, a dominant-negative mutant of cJun, antagonizes the Jun-mediated transcriptional repression. (a) KGN cells were co-transfected with 250 ng of various expression vectors and either the aromatase PII or a synthetic AP1-responsive luciferase reporter construct. Cells in the left panel were treated with or without forskolin (25 mM) overnight before harvest. (b) KGN cells were co- transfected with the aromatase PII-luciferase construct and increasing amounts of the cJun expression vector, along with either a fixed amount of TAM-67 (250 ng; solid bars) or pcDNA3 (250 ng; open bars). Cells were treated with forskolin (25 mM) overnight before subject to luciferase assay. Luciferase data shown are average of results from triplicates

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Dominant Negative Mutation, Transfection, Expressing, Luciferase, Construct, Plasmid Preparation

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 6 Both the AD and bZIP domains of JunB are required for the Jun-mediated transcriptional repression. (a) A schematic diagram depicting the locations of the various functional domains in JunB and the positions of point mutations. (b) KGN cells were co-transfected with the aromatase PII-luciferase construct along with the wild-type or various mutant JunB expression vectors. To achieve equivalent protein expression levels, the following amounts of plasmids were used: 100 ng FL, 250 ng KCID and LG; and 500 ng JunB-C. Transfected cells were treated overnight with forskolin (25 mM). Expression of the wild-type and mutant JunB proteins is shown in the inset. FL: full-length JunB; KCID: JunB(K284I/C285D); LG: JunB(L303G); BC: JunB-C terminus. Luciferase data shown are average of results from triplicates

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Functional Assay, Transfection, Luciferase, Construct, Mutagenesis, Expressing

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 7 Determination of the cis-acting elements in the aromatase PII promoter that mediate the transcription repression by Jun. (a) Schematic diagram of the ovary-specific aromatase promoter. Also indicated are the known regulatory sequences, including the CLS, binding site for steroidogenic factor 1 (SF-1), and TATA box. (b) KGN cells were co-transfected with various 50-promoter deletion constructs with or without JunB expression construct (250 ng). At 24 h after transfection, cells without ectopic JunB were incubated overnight in the presence (solid bars; 2, 5, 8, 11, 14, and 17) or absence (gray bars; 1, 4, 7, 10, 13, and 16) of forskolin. All samples with ectopic JunB expression were treated with forskolin (open bars; 3, 6, 9, 12, 15, and 18). Luciferase data shown are average of results from triplicates

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Binding Assay, Transfection, Construct, Expressing, Incubation, Luciferase

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 8 Endogenous Jun proteins were associated with the CLS element. (a) EMSA was performed with a 32P-labeled oligonucleo- tide probe corresponding to the CLS element in the aromatase PII promoter. Nuclear extracts were prepared from the KGN granulosa cells treated with or without forskolin (25 mM) and were incubated with the radioactive probe in the presence and absence of different antibodies. The sample in lane 1 only contained buffer and the free probe. Rabbit IgG was used as a negative control (lane 2). Protein–DNA complexes are marked as I, II and III. The super- shift bands caused by anti-cJun and anti-JunD antibodies are indicated by asterisks. Similar results were obtained from four independent experiments. (b) ChIP was carried out with KGN cells. Either rabbit IgG or an anti-cJun antibody was used to precipitate the crosslinked chromatin fragments. Semiquantitative PCR was conducted to assess the relative amount of promoter-proximal versus distal (2 kb upstream) regions in the immunoprecipitates. The relative positions of the primers sets in the PII promoter are indicated in the top diagram. The result shown is a representative of three independent ChIP experiments

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Labeling, Incubation, Negative Control

Journal: Oncogene

Article Title: Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element.

doi: 10.1038/sj.onc.1208415

Figure Lengend Snippet: Figure 9 The CLS element is required for the Jun-mediated repression of the aromatase PII promoter. KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b). Cells were treated with (solid bars) or without (open bars) forskolin (25 mM) overnight

Article Snippet: KGN cells were co-transfected with an increasing amount of the JunB expression vector and either the wild-type PII-luciferase reporter (a) or a mutant version in which the CLS element was replaced by a GAL4-binding site (b).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Mutagenesis, Binding Assay

Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: YAP1 interacts with EGFR signaling pathway to drive granulosa cell proliferation. A) Knockdown of Yap1 in mouse granulosa cells reduced mRNA levels of Egfr, EGF-like ligands (Hbegf and Areg), and cyclin D1 (Ccnd1). Relative mRNA levels were determined using RT-PCR. The experiment was repeated ≥3 times, and representative images are presented. B) Expression of HBEGF in hGCs, KGN cells, and HGrC1 cells transfected with empty vectors [MX as control (CTL)], vectors expressing wild-type YAP1 (YAP), or constitutively active YAP1 (YAPS127A). Relative mRNA levels were quantified using real-time PCR. Each bar represents the mean ± sem (n = 4). C, D) HBEGF treatment (50 ng/ml, 3 d) stimulated cell proliferation (C), but suppressed estradiol (E2) and progesterone (P4) production (D) in cultured granulosa cells. Each bar represents the mean ± sem (n = 4). E) HBEGF (50 ng/ml) induced rapid dephosphorylation of LATS1 (S909) and YAP1 (S127) and phosphorylation of ERK1/2 in cultured granulosa cells. β-Actin protein was used as loading CTL. The relative levels of the total proteins and phosphoproteins were determined by Western blot. Ctgf, connective tissue growth factor; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; p, phosphorylated. Experiments were repeated ≥3 times, and representative images are presented. *P < 0.05, **P < 0.01, ***P < 0.001, compared with corresponding CTL groups (MX groups).

Article Snippet: HGrC1 [an immortalized human granulosa cell (hGC) line] and KGN (an ovarian granulosa cell–like tumor cell line) cells were obtained from the Riken Cell Bank (Riken BioResource Research Center, Tsukuba, Japan).

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Control, Real-time Polymerase Chain Reaction, Cell Culture, De-Phosphorylation Assay, Phospho-proteomics, Western Blot

Journal: The FASEB Journal

Article Title: Timely expression and activation of YAP1 in granulosa cells is essential for ovarian follicle development

doi: 10.1096/fj.201900179RR

Figure Lengend Snippet: YAP1 stimulates proliferation but suppresses differentiation of granulosa cells. A) Representative images showing spheroids formed in KGN-MX, KGN-YAP, and KGN-YAPS127A cells in a 3D culture system. Scale bars, 200 μm. B) Numbers of spheroids with different diameters formed by KGN-MX, KGN-YAP, and KGN-YAPS127A cells in the 3D culture system in A. Each bar represents the mean ± sem (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with corresponding control (CTL). C) Production of E2 and progesterone in KGN-MX, KGN-YAP, and KGN-YAPS127A cells in the 3D culture system. *P < 0.05, **P < 0.01, ***P < 0.001 compared with MX control. D) Knockdown of Yap1 with Yap1-specific siRNA (siYap) in cultured mouse granulosa cells up-regulated the expression of granulosa cell differentiation–associated genes. The relative mRNA levels of Star, Fshr, Sult1e1, Ptgfr, cyp11A1, cyp19a1, and Gapdh were determined by RT-PCR. E) Forskolin (FSK) induced expression of differentiation-associated genes in cultured primary mouse granulosa cells. The relative mRNA levels of Yap1, Star, Fshr, Sult1e1, Ptgfr, cyp11a1, and cyp19a1 in mouse granulosa cells treated with 10 μM FSK for the indicated time points were determined by quantitative PCR. *P < 0.05, **P < 0.01, ***P < 0.001 compared with CTL. F) FSK (10 μM) induced phosphorylation of YAP1 in cultured HGrC1 cells. Please note that phosphorylation results in inactivation of YAP1 protein. The relative levels of the total proteins and phosphoproteins were determined by Western blot. All experiments were repeated ≥3 times, and representative images were presented. G) FSK (10 μM) suppressed the expression of Hbegf mRNA in cultured primary mouse granulosa cells in a time-dependent manner. Each bar represents the mean ± sem (n = 4). *P < 0.05, **P < 0.01 compared with CTL. H) FSK (10 μM, 72 h) suppressed HBEGF mRNA expression in cultured primary hGCs. CREB, cAMP responsive element binding protein; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; p, phosphorylated. Each bar represents the mean ± sem (n = 4). **P < 0.01 compared with CTL.

Article Snippet: HGrC1 [an immortalized human granulosa cell (hGC) line] and KGN (an ovarian granulosa cell–like tumor cell line) cells were obtained from the Riken Cell Bank (Riken BioResource Research Center, Tsukuba, Japan).

Techniques: Control, Knockdown, Cell Culture, Expressing, Cell Differentiation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Binding Assay

Journal: Journal of Ovarian Research

Article Title: Integrating network pharmacology and experimental verification to explore the pharmacological mechanisms of asparagus against polycystic ovary syndrome

doi: 10.1186/s13048-023-01210-5

Figure Lengend Snippet: ASP reverses DHEA induced decrease of PRKCA in PCOS granulosa cells. A - C qRT-PCR and Western blot were performed to detect the expression of PRKCA in KGN cells. β-actin was used as a normalization standard. D Estrus status of mice in the DHEA and control groups (n = 6 per group). E ELISA assay for T and E2 in mouse serum. F Histological staining of ovaries from DHEA and control mice. G qRT-PCR was performed to detect the expression of PRKCA in PCOS mouse models

Article Snippet: KGN cells, a steroidogenic human granulosa cell-like tumor cell line was purchased from iCell Bioscience Inc (China) and identified by short tandem repeat (STR) profiling.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: microRNA-194 is increased in polycystic ovary syndrome granulosa cell and induce KGN cells apoptosis by direct targeting heparin-binding EGF-like growth factor

doi: 10.1186/s12958-021-00850-w

Figure Lengend Snippet: miR-194 regulated KGN cell growth and apoptosis. ( A ) A CCK-8 assay assessed KGN cell viability upon treatment with miR-194 mimic or inhibitor. ( B ) Ki67 was utilized to determine the proliferative ability of KGN cells upon transfection with miR-194 or miR-NC inhibitors. ( C - D ) The AO/EB and flow cytometry were used to detect the KGN cell apoptosis. ( E ) p53, p21 and p16 protein level after treatment with miR-194 or miR-NC mimic. 𝑛 = 3-6 for each group. * P < 0.05 vs. miR-NC group

Article Snippet: KGN cells were purchased from the Riken Cell Bank (Wako, Saitama, Japan) [ ].

Techniques: CCK-8 Assay, Transfection, Flow Cytometry

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: microRNA-194 is increased in polycystic ovary syndrome granulosa cell and induce KGN cells apoptosis by direct targeting heparin-binding EGF-like growth factor

doi: 10.1186/s12958-021-00850-w

Figure Lengend Snippet: HB-EGF was directly targeted by miR-194. A Venn diagram demonstrates the intersection of data extracted from several bioinformatics websites. ( B ) Binding sites shared between miR-194 and HB-EGF. ( C - D ) HB-EGF protein and mRNA expression in the GCs. KGN cells were transfected for 48 h with miR-194 mimic, miR-194 inhibitor, or the miR-NC group. ( E ) Luciferase reporter assays. ( F ) miR-194 and HB-EGF mRNA expression in PCOS patient ovarian GCs. n= 3-6 for each group; *P < 0.05 vs. control group or miR-NC group

Article Snippet: KGN cells were purchased from the Riken Cell Bank (Wako, Saitama, Japan) [ ].

Techniques: Binding Assay, Expressing, Transfection, Luciferase

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: microRNA-194 is increased in polycystic ovary syndrome granulosa cell and induce KGN cells apoptosis by direct targeting heparin-binding EGF-like growth factor

doi: 10.1186/s12958-021-00850-w

Figure Lengend Snippet: Upregulated HB-EGF reverses the effect of miR-194 mimic in KGN cells growth and apoptosis. ( A ) The expression of HB-EGF in KGN cells post-transfection with HB-EGF-OE. ( B ) The expression of HB-EGF in KGN cells post transfection with miR-194 mimic or miR-194 mimic+HB-EGF-OE. ( C - D ) The apoptotic rate of KGN cells was determined using AO/EB and flow cytometry. 𝑛 = 3-6 for each group; * P < 0.05 vs. miR-NC mimic group

Article Snippet: KGN cells were purchased from the Riken Cell Bank (Wako, Saitama, Japan) [ ].

Techniques: Expressing, Transfection, Flow Cytometry